Polymerase Slippage Restoration of Frameshifted TGFBR2 in Colorectal Cancer: A Novel Paradigm

Abstract

See “Transforming growth factor β signaling in colorectal cancer cells with microsatellite instability despite biallelic mutations in TGFBR2,” by de Miranda NFCC, van Dinther M, van den Akker BEWM, et al, on page 1427. See “Transforming growth factor β signaling in colorectal cancer cells with microsatellite instability despite biallelic mutations in TGFBR2,” by de Miranda NFCC, van Dinther M, van den Akker BEWM, et al, on page 1427. The transforming growth factor (TGF)-β signaling pathway consists of a family of secreted TGF-β–like proteins (the TGF-β isoforms [TGF-β1, TGF-β2, and TGF-β3], activins, and others), the TGF-β receptors, and a variety of post-receptor signaling proteins. It was discovered >30 years ago and has been since shown to have a role in a variety of diseases, including colorectal cancer (CRC).1Anzano M.A. Roberts A.B. Meyers C.A. et al.Synergistic interaction of two classes of transforming growth factors from murine sarcoma cells.Cancer Res. 1982; 42: 4776-4778PubMed Google Scholar These 3 decades of study have led to an in-depth understanding of TGF-β signaling deregulation in CRC. However, despite our detailed understanding of TGF-β signaling in CRC, in this issue of Gastroenterology, de Miranda et al show us that there remains much to learn.2de Miranda N.F.C.C. van Dinther M. van den Akker B.E.W.M. et al.Transforming growth factor β signaling in colorectal cancer cells with microsatellite instability despite biallelic mutations in TGFBR2.Gastroenterology. 2015; 148: 1427-1437Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar To appreciate the significance of the findings by de Miranda, it is important to understand some key aspects of TGF-β signaling. TGF-β mediates its effects on cells through a heteromeric TGF-β receptor complex that consists of type I (TGFBR1) and type II (TGFBR2) components. TGFBR1 and TGFBR2 are cell membrane–associated, single-pass transmembrane serine-threonine kinases that phosphorylate downstream signaling proteins upon activation and are the only known receptor complex for TGF-β.3Massague J. TGF-β signaling: receptors, transducers, and mad proteins.Cell. 1996; 85: 947-950Abstract Full Text Full Text PDF PubMed Scopus (826) Google Scholar After becoming activated by TGF-β, TGFBR2 phosphorylates TGFBR1 in the GS box region, which activates TGFBR1. TGFBR1 then propagates the signal from the receptor to the nucleus through the phosphorylation of downstream proteins, including the SMAD proteins (SMAD2 and SMAD3) and non-SMAD proteins (including PI3K, p38MAPK, PKA, and RhoA).4Markowitz S. Roberts A. Tumor suppressor activity of the TGF-β pathway in human cancers.Cytokine and Growth Factor Reviews. 1996; 7: 93-102Crossref PubMed Scopus (396) Google Scholar, 5Wakefield L.M. Roberts A.B. TGF-beta signaling: positive and negative effects on tumorigenesis.Curr Opin Genet Dev. 2002; 12: 22-29Crossref PubMed Scopus (729) Google Scholar, 6Chowdhury S. Howell G.M. Rajput A. et al.Identification of a novel TGFbeta/PKA signaling transduceome in mediating control of cell survival and metastasis in colon cancer.PLoS One. 2011; 6: e19335Crossref PubMed Scopus (42) Google Scholar, 7Zhang Y.E. Non-SMAD pathways in TGF-beta signaling.Cell Res. 2009; 19: 128-139Crossref PubMed Scopus (1316) Google Scholar In epithelial cells, including those in the intestine, TGF-β can inhibit cell proliferation; induce apoptosis, senescence, and terminal differentiation; and maintain genomic stability, which has led to the conclusion that this pathway has tumor suppressor activities in cancers, including CRC. Indeed, a large body of evidence from in vitro, in vivo, and human studies has established that the TGF-β signaling pathway has a prominent role as a tumor suppressor pathway in CRC. Members of the TGF-β pathway, including TGFBR1, TGFBR2, SMAD2, SMAD4, and others, are commonly mutated or silenced in CRC, making this pathway the most commonly altered pathway in CRC.8Cancer Genome Atlas NetworkComprehensive molecular characterization of human colon and rectal cancer.Nature. 2012; 487: 330-337Crossref PubMed Scopus (5931) Google Scholar Overall, 20%-30% of CRCs have TGFBR2 mutations, and in microsatellite unstable (MSI) CRCs, approximately 90% of tumors have inactivating mutations in TGFBR2, which occur in a 10-bp polyadenine tract in exon 3, called BAT-R2.9Markowitz S. Wang J. Myeroff L. et al.Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability.Science. 1995; 268: 1336-1338Crossref PubMed Scopus (2143) Google Scholar, 10Grady W.M. Myeroff L.L. Swinler S.E. et al.Mutational inactivation of transforming growth factor beta receptor type II in microsatellite stable colon cancers.Cancer Res. 1999; 59: 320-324PubMed Google Scholar, 11Myeroff L. Parsons R. Kim S.-J. et al.A TGF-beta receptor type II gene mutation common in colon and gastric but rare in endometrial cancers with microsatellite instability.Cancer Res. 1995; 55: 5545-5547PubMed Google Scholar However, despite the substantial amount of data demonstrating the tumor suppressor effects of TGF-β, the story of TGF-β signaling in CRC seems to be more complex than this pathway simply being a CRC tumor suppressor pathway. One twist in the TGF-β story is that TGF-β signaling can be oncogenic as well as tumor suppressive, acting as a molecular Dr. Jekyll and Mr. Hyde. Indeed, recent studies have suggested that, in certain contexts, TGF-β may promote the invasive or metastatic behavior of established cancer cells, suggesting that TGF-β has a paradoxical role in primary human cancers.12Bierie B. Moses H.L. Tumour microenvironment: TGFbeta: the molecular Jekyll and Hyde of cancer.Nat Rev Cancer. 2006; 6: 506-520Crossref PubMed Scopus (1098) Google Scholar This paradoxical behavior is believed to arise most commonly in late-stage cancers, but the timing of the activity change and the mechanisms underlying the switch of TGF-β from tumor suppressor to oncogene are only partly understood at this time.12Bierie B. Moses H.L. Tumour microenvironment: TGFbeta: the molecular Jekyll and Hyde of cancer.Nat Rev Cancer. 2006; 6: 506-520Crossref PubMed Scopus (1098) Google Scholar, 13Morrison C.D. Parvani J.G. Schiemann W.P. The relevance of the TGF-beta paradox to EMT-MET programs.Cancer Lett. 2013; 341: 30-40Crossref PubMed Scopus (159) Google Scholar Another wrinkle in the TGF-β story is that some CRCs with inactivating mutations in TGFBR2, which is required for TGF-β signaling, still seem to respond to TGF-β. The reports showing retained TGF-β signaling activity in CRC cell lines with mutant TGFBR2 are in the minority, but they raise the possibility that there are either non–TGF-β receptor pathways through which TGF-β can induce its effects, which is unlikely given the wealth of data showing TGFBR2 is required for TGF-β signaling in epithelial cells, or that certain mutant TGFBR2 have retained activity. Because the majority of TGFBR2 mutations in CRCs are frameshift mutations in BAT-R2, which results in deletion of the transmembrane domain and intracellular kinase domain of the receptor, the idea that mutant TGFBR2-BAT-R2 could generate a functional TGF-β receptor has been viewed as highly unlikely. However, in this issue of Gastroenterology, de Miranda et al provide convincing evidence that we may have been too quick to rule out the possibility that mutant TGFBR2 can generate functional TGFBR2. Their studies of mutant TGFBR2-BAT-R2 arose from their surprising observation that MSI CRCs often have activated SMAD signaling, even when these cancers carry mutant TGFBR2-BAT-R2 (and mutant ACVR2, which is an activin receptor that also can activate SMAD signaling by phosphorylating SMAD2). To determine how the TGF-β pathway could be active in CRCs with mutant TGFBR2, they carried out a clever and careful set of studies that demonstrated that functional TGFBR2 can be produced from mutant TGFBR2-BAT-R2 as a consequence of RNA polymerase slippage, which is a phenomenon that occurs when a polymerase mistakenly adds or deletes basepairs in a strand of RNA being synthesized. They have found that RNA polymerase slippage during transcription of mutant TGFBR2-BATR2 can cause wild-type TGFBR2 messenger RNA (mRNA) to be produced. Their demonstration of RNA polymerase bypass of frameshifting InDel mutations in human CRC is among the first demonstrations of this phenomenon in humans and, perhaps more important, in cancer.14Rockah-Shmuel L. Toth-Petroczy A. Sela A. et al.Correlated occurrence and bypass of frame-shifting insertion-deletions (InDels) to give functional proteins.PLoS Genet. 2013; 9: e1003882Crossref PubMed Scopus (35) Google Scholar, 15Miyadera K. Brierley I. Aguirre-Hernandez J. et al.Multiple mechanisms contribute to leakiness of a frameshift mutation in canine cone-rod dystrophy.PLoS One. 2012; 7: e51598Crossref PubMed Scopus (7) Google Scholar If their findings are true for other genes frequently affected by microsatellite mutations, such as PTEN and ACVR2, this study will redefine our understanding of the biological and possibly clinical effects of this class of mutations in MSI CRC and other MSI cancers. Over a decade ago, Derynck and Feng raised the possibility of a receptor threshold as the mechanism that determines the specificity of the effects of TGF-β. This model proposes that there is a critical expression/activation level of the TGF-β receptor that determines the specific responses of a cell to TGF-β.16Derynck R. Feng X.-H. TGF-β receptor signaling.Biochim Biophys Acta. 1997; 1333: F105-F150Crossref PubMed Scopus (508) Google Scholar, 17Fafeur V. O'Hara B. Bohlen P. A glycosylation-deficient endothelial cell mutant with modified responses to transforming growth factor-beta and other growth inhibitory cytokines: evidence for multiple growth inhibitory signal transduction pathways.Mol Biol Cell. 1993; 4: 135-144Crossref PubMed Scopus (27) Google Scholar, 18Rojas A. Padidam M. Cress D. et al.TGF-beta receptor levels regulate the specificity of signaling pathway activation and biological effects of TGF-beta.Biochim Biophys Acta. 2009; 1793: 1165-1173Crossref PubMed Scopus (74) Google Scholar This model suggests that the oncogenic or tumor suppressive effects of TGF-β in cancers is at least partly regulated by TGF-β receptor expression levels.19Bierie B. Moses H.L. TGF-beta and cancer.Cytokine Growth Factor Rev. 2006; 17: 29-40Crossref PubMed Scopus (362) Google Scholar Thus, these differential effects of “high” and “low” levels of TGF-β signaling pathway activation have been proposed to be a mechanism for the paradoxical effects of TGF-β on cancer cells. Furthermore, it seems likely that the SMAD pathway and non-SMAD pathways are activated by different levels of TGF-β receptor activation, with the non-SMAD pathways requiring less TGF-β receptor activation to be induced.5Wakefield L.M. Roberts A.B. TGF-beta signaling: positive and negative effects on tumorigenesis.Curr Opin Genet Dev. 2002; 12: 22-29Crossref PubMed Scopus (729) Google Scholar, 20Rojas A. Padidam M. Cress D. et al.TGF-beta receptor levels regulated the specificity of signaling pathway activation and biological effects of TGF-beta.Biochim Biophys Acta. 2009; 1793: 1165-1173Crossref PubMed Scopus (78) Google Scholar This difference in pathway activation is relevant to CRCs because the SMAD pathway may be predominantly a tumor suppressor pathway and the non-SMAD pathways may be primarily oncogenic.5Wakefield L.M. Roberts A.B. TGF-beta signaling: positive and negative effects on tumorigenesis.Curr Opin Genet Dev. 2002; 12: 22-29Crossref PubMed Scopus (729) Google Scholar This aspect of TGF-β signaling is germane to the findings by de Miranda et al, because they have found that wild-type TGFBR2 mRNA produced from mutant TGFBR2-BAT-R2 is present at very low levels. They showed that it was expressed at a high enough level to activate the SMAD pathway but have not characterized the non-SMAD pathways, which would be predicted to also be active in these CRCs.20Rojas A. Padidam M. Cress D. et al.TGF-beta receptor levels regulated the specificity of signaling pathway activation and biological effects of TGF-beta.Biochim Biophys Acta. 2009; 1793: 1165-1173Crossref PubMed Scopus (78) Google Scholar If such a phenomenon is occurring in CRCs with mutant TGFBR2-BAT-R2, then the BAT-R2 mutations may be inducing oncogenic activity in the CRC cells, providing an unrecognized mechanism through which TGFBR2 mutations may promote CRC formation. However, despite these provocative implications, an important limitation of De Miranda’s study is that a clear biological effect of TGF-β was not evident in the CRCs with mutant BAT-R2. So, it remains to be proven that the mutant TGFBR2 produces enough functional TGFBR2 to have an obvious effect on CRC cell behavior. The demonstration of RNA polymerase slippage in CRC is an aspect of the study by de Miranda et al that deserves special attention. DNA and RNA polymerase slippage occur when a polymerase inserts or deleted basepairs in error, and often is seen in repetitive sequences of basepairs, such as polyadenine tracts (Figure 1). Although often thought of in the context of generating faulty transcripts, de Miranda et al observed slippage of RNA polymerase in MSI CRCs that resulted in RNA editing and restoration of wild-type TGFBR2 mRNA sequence in those CRCs with 1-bp deletions in the TGFBR2-BAT-R2 DNA. Polymerase slippage has been observed in model organisms and in other contexts, but the findings in this study demonstrate that this can also occur in human cancer cells.15Miyadera K. Brierley I. Aguirre-Hernandez J. et al.Multiple mechanisms contribute to leakiness of a frameshift mutation in canine cone-rod dystrophy.PLoS One. 2012; 7: e51598Crossref PubMed Scopus (7) Google Scholar, 21Paoloni-Giacobino A. Rossier C. Papasavvas M.P. et al.Frequency of replication/transcription errors in (A)/(T) runs of human genes.Hum Genet. 2001; 109: 40-47Crossref PubMed Scopus (24) Google Scholar Although further study is needed, if this phenomenon is shown for other genes with microsatellite repeat mutations, de Miranda et al may have discovered a mechanism through which mutations can regulate gene activity in a graded rather than binary fashion. In summary, their findings are notable not only because have they found a reason for the retained TGF-β activity observed in some MSI CRCs with inactivating mutations in TGFBR2, but also because they have provided important and potentially paradigm shifting insight our understanding of frameshift mutations in cancer. Transforming Growth Factor β Signaling in Colorectal Cancer Cells With Microsatellite Instability Despite Biallelic Mutations in TGFBR2GastroenterologyVol. 148Issue 7PreviewMost colorectal cancer (CRC) cells with high levels of microsatellite instability (MSI-H) accumulate mutations at a microsatellite sequence in the gene encoding transforming growth factor β receptor II (TGFBR2). TGFβ signaling therefore is believed to be defective in these tumors, although CRC cells with TGFBR2 mutations have been reported to remain sensitive to TGFβ. We investigated how TGFβ signaling might continue in MSI-H CRC cells. Full-Text PDF Covering the CoverGastroenterologyVol. 148Issue 7PreviewThe use of biological agents has been a major advance in the treatment of patients with inflammatory bowel disease. For example, weight-based dosing (5 mg/kg) of the chimeric immunoglobulin (Ig)G1 monoclonal anti-tumor necrosis factor antibody, infliximab, involving an induction phase followed by maintenance treatment has been shown to be effective in inducing and maintaining clinical remission in patients with Crohn’s disease (CD) and ulcerative colitis (UC). However, over time, therapeutic benefit is lost in many patients, requiring a dose escalation or a switch to another biological agent. Full-Text PDF